Oxidative stress has been implicated as a factor in various diseases and injury states. However, the extent to which oxidative stress could be predictive of clinical outcome was unknown, nor was a satisfactory method for rapidly assaying total oxidative stress in a subject available.
Various methods of measuring particular oxidants within biological fluids are known. However, these methods generally rely on the measurement of only a single oxidized compound, or a narrow class of compounds, as indicators of oxidative activity. Thus, they may not be reliable predictors of overall oxidative stress. For example, Draper et al. (Lipids 19:836-843, 1984) disclose the use of urinary malondialdehyde as an indicator of lipid peroxidation. These approaches may be limited to the measurement of lipid peroxide breakdown products, and may not adequately reflect oxidation by hydrogen peroxide and organic peroxides.
Attempts have been made to measure oxidative stress through the measurement of hydrogen peroxide in urine (Long et al., BBRC, 262,605-609 (1999)). However, these approaches are limited to the measurement of hydrogen-peroxide derived oxidative species, and may not provide an accurate assessment of overall oxidative stress.
A method of assaying hydrogen peroxide in fluids using 2-deoxyribose was developed by Halliwell, et al. (Analytical Biochemistry, 165,215-219 (1987)). Hydroxyl radicals were generated by the reaction of an Fe3+ EDTA complex with hydrogen peroxide in the presence of ascorbic acid. These hydroxyl radicals then attacked the deoxyribose to form products which, upon heating with thiobarbituric acid (“TBA”) at low pH yield a pink chromogen. The chromogen could then be measured to provide an estimate of the original hydrogen peroxide concentration. However, Halliwell's method is adapted for the measurement of hydroxyl radical, and no consideration was given to the measurement of overall oxidative stress from a range of reactive species. Moreover, the use of ascorbic acid in Halliwell's method renders the reagent mixture relatively unstable, and may render it unsuitable for use in routine clinical testing, and other applications where stability is important. A further disadvantage of Halliwell's method is that the reaction is slow, and of limited accuracy, which may make it unsuitable for use in situations where rapid or precise results are needed.
Thus, it is an object of the present invention to provide a rapid assay for the measurement of overall oxidative stress.